1. Technical Field
The present invention relates to a method and device for producing a vaccine. More specifically, the present invention relates to a protein-free cell culture process for producing a vaccine.
2. Description of the Related Art
Immunization to protect against communicable disease is one of the most successful and cost-effective practices of modern medicine. Smallpox has been completely eliminated by vaccination, and the incidence of many other dreaded diseases such as polio and diphtheria has been drastically reduced through immunization programs. However, vaccines, especially those based on the use of inactivated viruses, vary in effectiveness. For example, while the currently licensed influenza vaccine is reportedly over 80% efficacious in young adults, it is only approximately 60% efficacious in adults 65 years of age and older, and less than 50% effective in children under 2 years of age. The recently licensed chicken pox vaccine is reportedly approximately 70% efficacious, and there are currently no effective vaccines against many important viral diseases including those caused by respiratory syncytial virus, parainfluenza 3 virus, Rotavirus, and the human immunodeficiency virus. In some cases, licensed inactivated viral vaccines may cause adverse reactions that have prevented their use at the higher dosages needed to improve efficacy.
Inactivated virus vaccines confer protection by stimulating immune responses to proteins found in the free virus. Antibodies to the mature envelope proteins found on free virus may be optimal in blocking the initial events of infection (such as virus binding to a cell receptor and attachment and entry into a cell) following exposure to a virus, but may be sub-optimal once a virus has entered a cell. Once infected, the cells and the cell-associated immature virions contain precursors to the mature envelope proteins. These precursor proteins may stimulate more optimal immune responses for stemming the spread of infection and preventing clinical illness when the body's first line of defense, antibodies to free virus, does not completely prevent all viruses from infecting cells.
Inactivated virus vaccines are typically produced from virus that has been grown in animal cells, e.g. embryonated eggs for influenza, which are then inactivated by treatment with chemicals such as formalin. Attenuated vaccines for measles and chickenpox are produced by growing weakened virus in cell cultures. Advances in the understanding of the pathogenesis of viral infections and recombinant DNA technology have led to the identification and production of specific viral proteins for use in subunit viral vaccines. These have been particularly successful in the formulation of a subunit vaccine against the hepatitis B virus.
Most existing licensed vaccines and vaccines in development, whether based on inactivated viruses or recombinant DNA technology, rely primarily on immune responses to the mature virus, or, in a few examples of experimental, recombinant DNA-based vaccines, immune responses to antigens found in the cell-associated form of the virus, or virus-infected cells. Both the killed virus and attenuated virus approaches on the one hand and the recombinant DNA approaches on the other hand have their advantages and their limitations. While the cell culture and embryonated egg methods are used to grow whole virus very inexpensively, they are not very efficient methods for the commercial production of the viral precursor proteins found in the infected cells and the cell-associated forms of the virus. This is because these methods act like miniature assembly lines and, while a large amount of mature virus accumulates in the cell cultures or the eggs at any given time, a much smaller amount of virus is actually in the process of being assembled. Therefore, the purified virus used to make the vaccine contains very little, if any, of the envelope precursor or other precursor proteins. On the other hand, viral membrane glycoproteins, in either their mature or precursor form, can be efficiently produced by recombinant DNA technology. When native conformational structure is needed to produce functional, neutralizing antibodies, the use of recombinant technology employing mammalian cell or insect cell substrates is preferred. However, production of viral vaccine proteins in insect or mammalian cells by recombinant methods is generally more expensive on a per milligram protein basis than cell culture and egg production methods.
Adverse reactions from vaccines may arise from impurities or from biologic properties of the vaccine proteins (antigens) responsible for conferring protective immunity. For example, the contaminating egg protein present in the licensed influenza vaccines may be largely responsible for the adverse reactions associated with these products.
Mature viral proteins present in vaccines may have biologic properties that are responsible for adverse reactions. Uptake by mononuclear cells and granulocytes of inactivated influenza virus mediated by the mature hemagglutinin may also be responsible for adverse reactions. The mature HIV envelope glycoprotein (gp120) in some experimental vaccines against HIV may bind to the CD4 receptor of T4 lymphocytes and alter normal immune function. It would be desirable to reduce potential adverse reactions in the vaccine preparations.
The viral envelope proteins in inactivated virus vaccines are substantially glycosylated. While glycosylation is important in maintaining conformational structure of these proteins it may also reduce their immunogenicity. These proteins in either the mature of precursor form can be produced with trimmed carbohydrate residues with recombinant baculovirus expression vectors and insect cells. The baculovirus-produced proteins retain sufficient native conformation to stimulate functional neutralizing antibodies and may provide greater immunogenicity than highly glycosylated native proteins.
Infection by influenza virus causes substantial illness and premature death worldwide. Immunization with vaccines comprised of preparations of inactivated influenza viruses is currently the most useful practice for reducing disease from viral influenza. The vaccines confer protection against infection and disease by stimulating the production of immune responses to the hemagglutinin (HA), neuraminidase (NA), nucleoproteins (NP, M1) and possibly other proteins of component strains (Murphy, B. R., et al., N. Engl. J. Med. 268:1329-1332 (1972) and Kendal, A. P., et al., J. Infect. Dis. 136:S415-24 (1986)). The most important of these is the production of neutralizing antibodies to HA (Ada, G. L., and Jones, P. D., Curr. Top. Microbiol. Immunol. 128:1-54 (1986)). The currently available inactivated vaccines nevertheless have limitations, including sub-optimal immunogenicity and efficacy in adults 65 years of age and older and very young children and under utilization in part due to poor patient acceptance in connection with the belief that such vaccines are not very effective and fears of adverse reactions (Nichol, K. L., et al., Arch. Int. Med. 152:106-110 (1992)). The perception of lack of effectiveness arises in part from variations in potency from year to year and the association of many non-influenza respiratory tract illnesses with influenza.
The mature influenza virus contains both HA and NA proteins in its outer envelope. The HA is present as trimers. Each HA monomer consists of two polypeptides (HA1 and HA2) linked by a disulfide bond. These polypeptides are derived by cleavage of a single precursor protein, HA0, during maturation of the influenza virus. In part, because these molecules are tightly folded, the HA0 and the mature HA1 and HA2 differ slightly in their conformation and antigenic characteristics. Furthermore, the HA0 is more stable and resistant to denaturation and to proteolysis. Recently it has been reported that a baculovirus/insect cell culture derived recombinant HA0 conferred protective immunity to influenza (Wilkinson, B., MicroGeneSys Recombinant Influenza Vaccine, PMA/CBER Viral Influenza Meeting, Dec. 8, 1994). One limitation of recombinant HA0 vaccines is their inability to stimulate immune responses against non-HA antigens that may provide greater and more durable protection, especially for high-risk populations that do not respond well to immunization.
Current production of human influenza vaccine takes place primarily in fertile chicken eggs. Worldwide several hundred million of eggs are used each year to produce vaccine for the influenza season. The current production cycle (beginning with identification of the anticipated virus strains expected to be present in the forthcoming influenza season) is many months long. The current production processes that use fertile eggs as tiny bioreactors is labor intensive, expensive and fraught with variables, such as the seasonal availability and variation of properties of the eggs.
It would therefore be desirable to provide improved virus vaccine preparations that do not exhibit as many of the limitations and drawbacks observed with the use of currently available vaccines.